Abstract

α-Synuclein (α-Syn) is a central player in the pathophysiology of dopaminergic neurodegeneration that occurs in Parkinson’s disease. Recently, it has been demonstrated that α-Syn is highly expressed in human melanoma cell line, SK-MEL28 but is undetectable in non-melanocytic cutaneous carcinoma and normal skin. Its wild-type form was also found to inhibit apoptosis in response to various pro-apoptotic stimuli, making it another attractive candidate in oncogenesis besides γ-Syn. Therefore, the objective of this study was to investigate the cyto-toxic/-protective roles of α-Syn in melanoma SK-MEL28 cells, by knockdown and subjecting cells to apoptotic stimulus, staurosporine. For knockdown, short hairpin RNAs (shRNAs) targeting α-Syn were constructed in the expression vector, pLKO.1 and then transfected into SK-MEL28 cells with pLKO.1-TRC control and pLKO.1-scramble shRNA as controls. Stably transfected cells were established by selection with puromycin and α-Syn protein expression was then confirmed by Western blotting. The effects of α-Syn knockdown on cell viability, morphology, expression of Bcl-2, Bcl-XL, Bax and cleaved caspase 9 and proliferative index after staurosporine treatment were studied. Cell viability MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay showed that the survival of SK-MEL28 with α-Syn knockdown was significantly reduced as compared to controls. Depletion of endogenous α-Syn was found to enhance staurosporine-induced cytotoxicity in SK-MEL28 with greater Bax/Bcl-2 and Bax/Bcl-xL ratios and cleaved caspase 9 level. α-Syn knockdown also reduced the proliferative index of SK-MEL28. Overall, these results suggest that the endogenously high α-Syn confers resistance against apoptosis in SK-MEL28 cells.